The chemiosmotic hypothesis predicts that in bacteria inward movement of hydrogen ions is coupled to ATP synthesis catalyzed by the membrane-bound ATPase (BFoF1). To test this idea directly, hydrogen ion entry into cells of Streptococcus lactis and Escherichia coli will be studied after imposition of an electrochemical potential difference for H ion (a "protonmotive force") of varying size. The membrane potential will be measured by K ion distributions in the presence of valinomycin; the pH gradient will be measured by distributions of weak acids (or bases), and from the amount of H ions which enters the cell. A study of these parameters in the presence and absence of ATP synthesis should give information about the coupling between ATP synthesis and H ions movements. Other experiments will focus on active transport of galactosides by the lactose transport system of E. coli. This system normally couples 1H ions/sugar during cotransport. Mutants will be isolated which use sodium rather than the hydrogen ion as the coupling cation. The chemical effect of cation binding on substrate interaction with the transport protein(s) can then be studied directly. Other mutants will be isolated in which there may be an altered stoichiometry of H ions/sugar coupling.